Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through F0 during ATP synthesis

Abstract

Rhodamine 123 (RH-123) was used to monitor the membrane potential of mitochondria isolated from rat liver. Mitochondrial energization induces quenching of RH-123 fluorescence and the rate of fluorescence decay is proportional to the mitochondrial membrane potential. Exploiting the kinetics of RH-123 fluorescence quenching in the presence of succinate and ADP, when protons are both pumped out of the matrix driven by the respiratory chain complexes and allowed to diffuse back into the matrix through ATP synthase during ATP synthesis, we could obtain an overall quenching rate proportional to the steady-state membrane potential under state 3 condition. We measured the kinetics of fluorescence quenching by adding succinate and ADP in the absence and presence of oligomycin, which abolishes the ADP-driven potential decrease due to the back-flow of protons through the ATP synthase channel, F(0). As expected, the initial rate of quenching was significantly increased in the presence of oligomycin, and conversely preincubation with subsaturating concentrations of the uncoupler carbonyl cyanide p-trifluoro-metoxyphenilhydrazone (FCCP) induced a decreased rate of quenching. N,N'-dicyclohexylcarbodiimide (DCCD) behaved similarly to oligomycin in increasing the rate of quenching. These findings indicate that RH-123 fluorescence quenching kinetics give reliable and sensitive evaluation of mitochondrial membrane potential, complementing steady-state fluorescence measurements, and provide a mean to study proton flow from the mitochondrial intermembrane space to the matrix through the F(0) channel.

Neuroprotective Effects of Bikaverin on H2O2-Induced Oxidative Stress Mediated Neuronal Damage in SH-SY5Y Cell Line

“…Rhodamine 123 (RH-123), a mitochondrial selective, cationic, and lipophilic dye, was used to monitor the membrane potential of mitochondria. Mitochondrial energization induces quenching of RH-123 fluorescence and the rate of fluorescence decay is proportional to the mitochondrial membrane potential (Baraccaa et al 2003). Thus, it is imperative to measure the fluorescence intensity of the probe after the application of a specific stimulus.…”

Mitochondrial quality control: Cell-type-dependent responses to pathological mutant mitochondrial DNA

“…Mitochondrial membrane potential (Dc m ) changes were evaluated by measuring rhodamine 123 fluorescence quenching 91,92 under the following conditions: 2 £ 10 6 cells were added to 0.5 ml buffer (250 mM sucrose, 10 mM HEPES, 100 AM K-EGTA, 2 mM MgCl 2 , 4 mM KH 2 PO 4 , pH 7.4) containing an ADP regenerating system (10 mM glucose and 2.5 U Hexokinase (Sigma-Aldrich, H5000)) and permeabilized with 20 mg/ml of digitonin (SigmaAldrich, D5628). Before rhodamine 123 (Molecular Probes ThermoFisher Scientific, R302) (50 nM) addition, samples were incubated with 1.8 mM malonate (Sigma-Aldrich, M4795), and 0.1 mM ADP (Sigma-Aldrich, A6646).…”

“…The oligomycin-sensitive ATP synthase activity in permeabilized cells was determined according as decribed. 91,92 Essentially, cybrids (2 £ 10 6 cells/ml) were incubated for 15 min with 60 mg/ml digitonin in a Tris-HCl buffer, pH 7.4. Complex I-driven ATP synthesis was induced by adding 10 mM:10 mM glutamate:malate (C 0.6 mM malonate (SigmaAldrich, M4795)) and 0.5 mM ADP (Sigma-Aldrich, A6646) to the sample.…”

Synergistic neurotoxicity of oxygen-glucose deprivation and tetrabromobisphenol A in vitro: role of oxidative stress

“…Depolarization of mitochondria results in the efflux of rhodamine from mitochondria, and its consequently increased concentration in the cytosol causes a rise in fluorescence [3]. CGC cultures were treated with rhodamine123 (10 µM) at 37°C for 30 min.…”